I. Field of the Invention
The present invention relates to a chemiluminescent assay by which chemiluminescent assay can be carried out efficiently and accurately. The chemiluminescent assay is especially useful for enzyme immunoassays.
II. Background of the Invention
Chemiluminescent assays enable detection and quantification of target substances in test samples quickly and highly sensitively, so that they are widely used in measuring viruses such as HIV and HCV, and other trace components in the body.
Among the chemiluminescent compounds, 1,2-dioxetanes are drawing attention as chemiluminescent substrates. Various improvements in sensitivity or ease of handling have been made for the chemiluminescent assays in which 1,2-dioxetanes as substrates are subjected to enzyme reactions and the emitted chemiluminescnece is measured.
Studies for promoting sensitivities of chemiluminescent assays have been vigorously made and various chemiluminescence enhancers which increase the intensity of the chemiluminescence so as to promote sensitivity of the assay have been discovered for the chemiluminescent assays using 1,2-dioxetanes as chemiluminescent substrates. A representative example of such a chemiluminescence enhancer is polyvinylbenzyltributylammonium chloride (hereinafter also referred to as xe2x80x9cTBQxe2x80x9d) (see Japanese Laid-open Patent Application (Kokai) No. 4-124185) and various improvements have been proposed for increasing the intensity of the chemiluminescence in the chemiluminescent assays using TBQ as a chemiluminescent enhancer.
In assay systems using such a chemiluminescence enhancer, when the enzyme concentration is low, the chemiluminescence-enhancing efficiency is extremely high, while when the enzyme concentration exceeds a certain level, the chemiluminescence-enhancing efficiency gradually decreases, so that the relationship between the enzyme concentration and the intensity of the emitted chemiluminescence is not linear or proportional. Therefore, it is difficult to use the calibration curve in the assays, so that measurements of enzyme concentrations could not be carried out efficiently and accurately. No countermeasures for improving the proportionality of the calibration curve have been proposed.
Accordingly, an object of the present invention is to provide a chemiluminescent assay in which the proportionality between the enzyme concentration and the intensity of the emitted chemiluminescence is increased.
The present inventors intensively studied to discover that the linearity or proportionality between the enzyme concentration and the intensity of the emitted chemiluminescence is increased by making a chemiluminescence intensity-adjusting agent co-exist in the reaction system, thereby completing the present invention.
That is, the present invention provides a chemiluminescent assay comprising subjecting a chemiluminescent substrate to an enzyme reaction in the presence of the enzyme carrying out the enzyme reaction, a chemiluminescence enhancer and a chemiluminescence intensity-adjusting agent; and measuring resulting chemiluminescence from reaction product.
By the present invention, the linearity or proportionality between the enzyme concentration and the intensity of the emitted chemiluminescence in chemiluminescent assays is increased. Therefore, chemiluminescent assays can be carried out more efficiently and more accurately than the known chemiluminescent assays.